Many microscopic imaging systems project a beam of light onto a sample, and generate images based on light received from the sample. In many imaging devices that scan a sample with an illumination beam (sometimes referred to herein as optical beam scanning systems), the amount of information that can be reliably gathered using such systems is often related to the shape and/or other physical characteristics of the beam. Various imaging systems utilize such a scanning illumination beam. For example, confocal microscopy systems focus a beam of light at a particular lateral location (e.g., in the x-y plane), and at a particular depth (e.g., in the z plane). By scanning a portion of the sample in two dimensions at various depths, data collected by the confocal microscopy system can be used to generate three-dimensional images that include subsurface features. Confocal microscopy systems are often used to scan ex vivo samples that can be placed on a stage, and the beam is scanned over the x-y plane parallel to the surface of the stage, and the beam is focused at various depths, for example, by changing the focal length of an optical system used to focus the beam on the sample.
In many imaging modalities that utilize optical beam scanning, the lateral resolution is related to the lateral size of the focused beam (i.e., how tightly the beam is focused), which imposes a limit on the system's ability to resolve small features in the direction perpendicular to the direction of travel of the beam. In such systems, the more tightly the beam is focused, the more resolving power the system generally has in the lateral direction. In general, as a beam is more tightly focused in the lateral direction the depth of focus (DOF) of the beam also becomes smaller. That is, it is difficult to create a beam with a tight focus laterally that is also relatively elongated in the depth direction. In general, in known devices such as cameras and projectors, the DOF is the difference between the farthest point from the lens (or other focusing optics) at which an image projected by the lens is considered in focus and the nearest point from the lens at which the image is considered in focus. For example, the imaging medium (e.g., image sensor or film) of a camera is within the DOF of the lens when the image captured by the imaging medium is in focus (note that this is assuming that the object(s) being imaged is within the depth of field of the optical system). Similarly, an image projected by a projector is within the DOF of the lens if the projected image is in focus. In such systems, one portion of an imaging medium or projection surface may be within the DOF while another part may be outside the DOF (e.g., because the imaging medium/screen may be tilted with respect to the optical axis). The DOF of a beam in an optical beam scanning an imaging system, however, is the range of depths over which features can be resolved by the imaging system. Accordingly, there is generally a relationship between the DOF of the beam and how tightly the beam is focused laterally. For example, as the beam becomes more tightly focused to increase lateral resolution, optical diffraction generally causes the beam to converge to the focal point, and to diverge from the focal point at larger angles, causing the range of depths over which features can be resolved to be smaller. Conversely, when the beam converges to the focal point at a shallower angle, the DOF increases, but this also generally results in lower lateral resolution.
In some imaging modalities, such as confocal microscopy, a small DOF is generally believed to be beneficial, as the system focuses the beam at various depths during scanning resulting in the ability to resolve smaller features in the depth direction. In such imaging systems, the axial resolution (e.g., the ability to resolve small objects in the direction aligned with the travel of the beam) is likely identical to the DOF, as the rapid loss of focus away from the focal plane prevents objects in other planes from spuriously contributing to the imaging of the focal plane itself. Accordingly, in such systems, a more tightly focused beam likely increases the maximum lateral resolution and axial resolution.
However, in other imaging modalities, such as optical coherence tomography (OCT), axial resolution is derived from the depth-ranging capability of the modality itself. That is, because OCT systems can simultaneously detect and separate signals from varying depths without changing the focal length of the beam optics, it is generally more desirable to use a beam with a large DOF, so that more depths may be simultaneously captured in a single pass. However, because of the relationship between the lateral tightness of the focus and DOF, high lateral resolution generally comes at the expense of DOF, and vice-versa. Conventional OCT techniques generally produce images of tissue reflectance with resolutions of approximately 10 micrometers (μm) axially and 30 μm laterally, which can generally facilitate visualization of microscopic architectural morphology, but is insufficient for resolving individual human cells, which are typically on the order of 10 μm in size.